mycobacteriophage ms6 lysa is a peptidoglycan amidase and a

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Mycobacteriophage Ms6 LysA: a Peptidoglycan Amidase and a Useful Analytical Tool Sebabrata Mahapatra, a Charles Piechota, a Filipa Gil, b Yufang Ma, a,c Hairong Huang, a,d Michael S. Scherman, a Victoria Jones, a Martin S. Pavelka, Jr., e Jose Moniz-Pereira, b Madalena Pimentel, b Michael R. McNeil, a Dean C. Crick a Department of Microbiology Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA a ; Centro de Patogénese Molecula, Unidade dos Retrovirus e Infecções Associadas, Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal b ; Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, People’s Republic of China c ; Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, People’s Republic of China d ; Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA e Since the peptidoglycan isolated from Mycobacterium spp. is refractory to commercially available murolytic enzymes, possibly due to the presence of various modifications found on this peptidoglycan, the utility of a mycobacteriophage-derived murolytic enzyme was assessed for an analysis of peptidoglycan from mycobacteria. We cloned, expressed, and purified the lysA gene prod- uct, a protein with homology to known peptidoglycan-degrading amidases, from bacteriophage Ms6. The recombinant protein was shown to cleave the bond between L-Ala and D-muramic acid of muramyl pentapeptide and to release up to 70% of the di- aminopimelic acid present in the isolated mycobacterial cell wall. In contrast to lysozyme, which, in culture, inhibits the growth of both Mycobacterium smegmatis and Mycobacterium tuberculosis, LysA had no effect on the growth of either species. However, the enzyme is useful for solubilizing the peptide chains of isolated mycobacterial peptidoglycan for analysis. The data indicate that the stem peptides from M. smegmatis are heavily amidated, containing few free carboxylic acids, regardless of the cross- linking status. M ycobacterium tuberculosis is a leading cause of disease-related deaths worldwide and is responsible for nearly 2 million deaths each year. Much of the pathology and general drug resis- tance that this pathogen demonstrates is believed to be related to its unique cell wall core, which consists of a peptidoglycan layer covalently attached to a mycolic acid layer via the polysaccharide arabinogalactan (1, 2). Although the overall structures of the pep- tidoglycans of M. tuberculosis and Mycobacterium smegmatis are currently reasonably well known, some important gaps in our understanding of the structure and synthesis of this macromole- cule remain. For example, it was recently shown that the carbox- ylic acid functions of the stem pentapeptide moiety of lipid II, a peptidoglycan precursor, are substantially amidated in M. smeg- matis (3) and other mycobacterial species (4). Hence, it is impor- tant to understand the nature, synthesis, and function of these modifications in mature peptidoglycan in M. smegmatis, M. tuber- culosis, and other Mycobacterium species. Typically, when analyzing the peptidoglycan structure, the peptidoglycan is chemically or enzymatically hydrolyzed to gen- erate soluble fragments that can be further analyzed (36). How- ever, chemical hydrolysis can result in the loss of features, and peptidoglycan isolated from Mycobacterium spp. is notoriously refractory to commercially available murolytic enzymes. This re- sistance to enzymatic hydrolysis is possibly due to the presence of some or all of the modifications found on mycobacterial pepti- doglycan, including an N-glycolyl function on the muramic acid, the amidation of carboxylic acids, and the addition of a glycine or serine to the peptide (5). These modifications are not unique to mycobacteria, as the amidation of carboxylic acid functions (7); N-glycolylation (8); and the addition of glycine (9), serine, or alanine (10, 11) have been reported for the peptidoglycans of other bacteria. Thus, an endolytic enzyme that is adapted to the hydrolysis of modified peptidoglycan could be a useful tool in the armamentarium of the mycobacteriologist and other researchers. Since peptidoglycan hydrolases are often specific to peptidoglycan cross-linking and secondary modifications (12), the murolytic ac- tivity of a mycobacteriophage endolysin was investigated. More than 220 mycobacteriophage genomes have been se- quenced, all of which are predicted to encode a putative endolysin (lysin A) (12). The protein encoded by lysA in mycobacterial phage Ms6 causes Escherichia coli cells to lyse, after the addition of CHCl 3 , when expressed in the heterologous host (13, 14). In ad- dition to Ms6 LysA, four other mycobacteriophages generate cleared zones in zymograms where lyophilized Micrococcus luteus was incorporated into the gel matrix (15, 1517), strongly suggest- ing that mycobacteriophage LysA proteins are murolytic. Thus, it seemed possible that LysA could be a useful reagent for generating peptidoglycan fragments from mycobacterial peptidoglycan for analytical purposes. This paper reports the expression, purifica- tion, and partial characterization of the enzymatic activity of LysA from Ms6 and the utilization of the enzyme in analyses of the amidation of mycobacterial peptidoglycan. It should be noted that the designation lysA is also used to identify an unrelated gene encoding an enzyme involved in diaminopimelic acid (DAP) syn- thesis; in this paper, lysA and LysA refer to the gene encoding the mycobacterial phage lysin and the lysin, respectively. Received 18 July 2012 Accepted 9 November 2012 Published ahead of print 16 November 2012 Address correspondence to Dean C. Crick, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.02263-12. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.02263-12 768 aem.asm.org Applied and Environmental Microbiology p. 768 –773 February 2013 Volume 79 Number 3 Downloaded from https://journals.asm.org/journal/aem on 25 November 2021 by 102.65.90.61.

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Page 1: Mycobacteriophage Ms6 LysA is a peptidoglycan amidase and a

Mycobacteriophage Ms6 LysA: a Peptidoglycan Amidase and a UsefulAnalytical Tool

Sebabrata Mahapatra,a Charles Piechota,a Filipa Gil,b Yufang Ma,a,c Hairong Huang,a,d Michael S. Scherman,a Victoria Jones,a

Martin S. Pavelka, Jr.,e Jose Moniz-Pereira,b Madalena Pimentel,b Michael R. McNeil,a Dean C. Cricka

Department of Microbiology Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USAa; Centro de Patogénese Molecula, Unidade dosRetrovirus e Infecções Associadas, Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugalb; Department of Biochemistry and Molecular Biology, Dalian MedicalUniversity, Dalian, People’s Republic of Chinac; Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, People’s Republic of Chinad; Department of Microbiology andImmunology, University of Rochester Medical Center, Rochester, New York, USAe

Since the peptidoglycan isolated from Mycobacterium spp. is refractory to commercially available murolytic enzymes, possiblydue to the presence of various modifications found on this peptidoglycan, the utility of a mycobacteriophage-derived murolyticenzyme was assessed for an analysis of peptidoglycan from mycobacteria. We cloned, expressed, and purified the lysA gene prod-uct, a protein with homology to known peptidoglycan-degrading amidases, from bacteriophage Ms6. The recombinant proteinwas shown to cleave the bond between L-Ala and D-muramic acid of muramyl pentapeptide and to release up to 70% of the di-aminopimelic acid present in the isolated mycobacterial cell wall. In contrast to lysozyme, which, in culture, inhibits the growthof both Mycobacterium smegmatis and Mycobacterium tuberculosis, LysA had no effect on the growth of either species. However,the enzyme is useful for solubilizing the peptide chains of isolated mycobacterial peptidoglycan for analysis. The data indicatethat the stem peptides from M. smegmatis are heavily amidated, containing few free carboxylic acids, regardless of the cross-linking status.

Mycobacterium tuberculosis is a leading cause of disease-relateddeaths worldwide and is responsible for nearly 2 million

deaths each year. Much of the pathology and general drug resis-tance that this pathogen demonstrates is believed to be related toits unique cell wall core, which consists of a peptidoglycan layercovalently attached to a mycolic acid layer via the polysaccharidearabinogalactan (1, 2). Although the overall structures of the pep-tidoglycans of M. tuberculosis and Mycobacterium smegmatis arecurrently reasonably well known, some important gaps in ourunderstanding of the structure and synthesis of this macromole-cule remain. For example, it was recently shown that the carbox-ylic acid functions of the stem pentapeptide moiety of lipid II, apeptidoglycan precursor, are substantially amidated in M. smeg-matis (3) and other mycobacterial species (4). Hence, it is impor-tant to understand the nature, synthesis, and function of thesemodifications in mature peptidoglycan in M. smegmatis, M. tuber-culosis, and other Mycobacterium species.

Typically, when analyzing the peptidoglycan structure, thepeptidoglycan is chemically or enzymatically hydrolyzed to gen-erate soluble fragments that can be further analyzed (3–6). How-ever, chemical hydrolysis can result in the loss of features, andpeptidoglycan isolated from Mycobacterium spp. is notoriouslyrefractory to commercially available murolytic enzymes. This re-sistance to enzymatic hydrolysis is possibly due to the presence ofsome or all of the modifications found on mycobacterial pepti-doglycan, including an N-glycolyl function on the muramic acid,the amidation of carboxylic acids, and the addition of a glycine orserine to the peptide (5). These modifications are not unique tomycobacteria, as the amidation of carboxylic acid functions (7);N-glycolylation (8); and the addition of glycine (9), serine, oralanine (10, 11) have been reported for the peptidoglycans ofother bacteria. Thus, an endolytic enzyme that is adapted to thehydrolysis of modified peptidoglycan could be a useful tool in thearmamentarium of the mycobacteriologist and other researchers.

Since peptidoglycan hydrolases are often specific to peptidoglycancross-linking and secondary modifications (12), the murolytic ac-tivity of a mycobacteriophage endolysin was investigated.

More than 220 mycobacteriophage genomes have been se-quenced, all of which are predicted to encode a putative endolysin(lysin A) (12). The protein encoded by lysA in mycobacterialphage Ms6 causes Escherichia coli cells to lyse, after the addition ofCHCl3, when expressed in the heterologous host (13, 14). In ad-dition to Ms6 LysA, four other mycobacteriophages generatecleared zones in zymograms where lyophilized Micrococcus luteuswas incorporated into the gel matrix (15, 15–17), strongly suggest-ing that mycobacteriophage LysA proteins are murolytic. Thus, itseemed possible that LysA could be a useful reagent for generatingpeptidoglycan fragments from mycobacterial peptidoglycan foranalytical purposes. This paper reports the expression, purifica-tion, and partial characterization of the enzymatic activity of LysAfrom Ms6 and the utilization of the enzyme in analyses of theamidation of mycobacterial peptidoglycan. It should be noted thatthe designation lysA is also used to identify an unrelated geneencoding an enzyme involved in diaminopimelic acid (DAP) syn-thesis; in this paper, lysA and LysA refer to the gene encoding themycobacterial phage lysin and the lysin, respectively.

Received 18 July 2012 Accepted 9 November 2012

Published ahead of print 16 November 2012

Address correspondence to Dean C. Crick, [email protected].

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02263-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.02263-12

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MATERIALS AND METHODSExpression of LysA. Ms6 LysA was readily expressed in a soluble formwith an N-terminal His tag by using the previously described plasmidpMG231 in E. coli M-15 pREP4 (Qiagen, Valencia, CA) cells (14). Theprotein was purified to near homogeneity by using His-Select HF nickelaffinity gel (Sigma, St. Louis, MO).

Preparation of [3H]diaminopimelic acid-labeled peptidoglycan ofM. smegmatis. A total of 50 �Ci of tritiated DAP (45 Ci/mmol; AmericanRadiochemicals, St. Louis, MO) was added to 250 ml of Middlebrook 7H9medium, without albumin, dextrose, and catalase (ADC) enrichment,supplemented with lysine at 40 �g/ml, which was then inoculated with M.smegmatis strain PM1482 (ept-1 �lysA4 rpsL6 �blaS �blaE) and incubatedat 37°C to the late log phase. The cells were centrifuged for 10 min at6,000 � g and resuspended in 20 mM Tris HCl (pH 7.9) containing 0.5 MNaCl and 20% glycerol (breaking buffer) at 4 ml/g of cell pellet. The cellswere then broken via six passes through a French pressure cell at 20,000lb/in2, and a cell wall-enriched pellet was recovered after centrifugation(40 min at 35,000 � g). The pellet was washed by resuspension in breakingbuffer and repeated centrifugation. Noncovalently attached proteins andcarbohydrates were removed by treatment in 2% SDS overnight at roomtemperature, and again, the pellet was isolated by centrifugation. Theresulting pellet was resuspended in 2% sodium dodecyl sulfate (SDS),brought to 100°C for 1 h, and recovered by centrifugation. Finally, thepellet was washed twice with water and then with 80% acetone, resultingin a purified mycolylarabinogalactan-peptidoglycan complex (MAPc)containing tritium-labeled DAP. The final preparation was resuspendedin water and stored at �80°C for future use.

Preparation of unlabeled and radiolabeled UDP-muramyl-penta-peptide and muramyl-pentapeptide. UDP-muramyl-pentapeptide(UDP-Mur-pentapeptide) was enzymatically synthesized as previouslydescribed (3). Separate preparations of UDP-Mur-[14C]pentapeptidewere made by incorporating L-[14C]Ala or D-[14C]Glu (PerkinElmer).Muramyl-pentapeptide (Mur-pentapeptide) and radiolabeled Mur-pen-tapeptides were prepared from UDP-Mur-pentapeptide and UDP-Mur-[14C]pentapeptide by hydrolysis in 0.2 M trifluoroacetic acid for 1 h at60°C. The samples were then dried and dissolved in water.

Treatment of [3H]DAP-labeled peptidoglycan with LysA. MAPcsamples containing radiolabeled peptidoglycan (2,600 cpm) were sus-pended in 100 �l of 50 mM ammonium acetate buffer at pH 5.0, 6.0, or7.0, and 40 �g of recombinant LysA was added. The mixtures were incu-bated overnight at room temperature with gentle rocking. Concentratedtrichloroacetic acid was added to a final concentration of 10%, and theresulting mixture was incubated on ice for 30 min before centrifugationfor 15 min at 14,000 � g. To determine the percentage of radioactivitysolubilized from the peptidoglycan, aliquots of the resulting supernatantsand pellets {after resuspension in 0.5% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS)} were ana-lyzed by liquid scintillation spectrometry.

Treatment of Mur-[14C]pentapeptide with LysA. Mur-[14C]penta-peptide samples, prepared as described above, containing radiolabeledpeptide were suspended in 100 �l of 50 mM ammonium acetate buffer atpH 5.0, and 40 �g of recombinant LysA was added. The mixtures wereincubated for the indicated times at room temperature with gentle rock-ing. Concentrated trichloroacetic acid was added to a final concentrationof 10%, and the resulting mixture was chilled on ice for 30 min beforecentrifugation for 15 min at 14,000 � g. Aliquots of the resulting super-natants were applied onto Kieselgel 60 F254 thin-layer chromatography(TLC) plates, which were then developed in isobutyric acid–1 M ammo-nium hydroxide (5:30, vol/vol), and the radioactive bands were visualizedby autoradiography.

Mass spectrometric analysis of peptides released from M. smegma-tis cell walls. Unlabeled MAPc was prepared and treated with LysA asdescribed above for the radiolabeled MAPc. The LysA-digested sampleswere clarified by centrifugation, and supernatants containing MAPc-de-rived peptides were deproteinated by ultrafiltration using Millipore

Ultrafree centrifugal ultrafiltration devices with a 5-kDa cutoff. The sam-ples were then subjected to liquid chromatography-mass spectrometry(LC-MS). An aliquot was applied onto a Phenomenex HyperClone ODSreverse-phase C18 column (5 �m [2.0 by 150 mm]) connected to anAgilent 1200 series high-performance liquid chromatography (HPLC)system, and the soluble Mur-peptides were eluted with a 0 to 80% lineargradient of methanol in 0.1% formic acid at a flow rate of 320 �l/min Theeluate was directly introduced into an Agilent 6250 quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an Agilent multimodesource operated in the simultaneous electrospray ionization and atmo-spheric pressure chemical ionization mode. The positive-ion MS and tan-dem MS (MS2) data were collected by using Agilent MassHunter work-station software and processed with the molecular feature extractoralgorithm (MFE) of Agilent MassHunter Qualitative Analysis software tofind molecular features (compounds with a defined exact mass and reten-tion time) present in each sample. A preset minimum abundance of 500counts was used to filter out low-abundance molecular features. A customdatabase containing calculated monoisotopic masses of possible mono-meric, dimeric, and trimeric peptides, including those with amidated car-boxylic acid residues and/or Gly residues (up to a molecular mass of 2kDa), was generated and used to identify the observed molecular features.The assigned identity of peptides was confirmed by MS2 when possible.

RESULTS AND DISCUSSIONExpression of LysA and its activity against isolated mycobacterialcell walls. Ms6 LysA (GenBank accession number AAG48318.1), a43,079-Da protein with a predicted amidase 2 domain, was readilyexpressed with an N-terminal His tag and purified to apparenthomogeneity, as assessed by SDS-polyacrylamide gel electropho-resis (data not shown), by immobilized nickel affinity chromatog-raphy. The ability of the enzyme to solubilize mycobacterial cellwalls was tested by using [3H]DAP-labeled MAPc from M. smeg-matis at pH 5.0, 6.0, or 7.0. Up to 70% of the radiolabeled DAPpresent in the MAPc could be solubilized by Ms6 LysA by diges-tion overnight at room temperature, which is consistent with thepreviously reported hydrolytic activity in zymograms (17). Therewas little difference in the amounts of material released at the threepH values tested, although 10 to 15% more radioactive materialwas consistently solubilized at pH 5.0 than at pH 7.0 (data notshown).

Determination of the mode of action of LysA. Initial experi-ments indicated that Ms6 LysA did not hydrolyze UDP-Mur-pen-tapeptide but did hydrolyze Mur-pentapeptide derived from theUDP-Mur-pentapeptide (data not shown). Thus, Mur-[14C]pen-tapeptide containing radiolabeled D-Glu residues was treated withenzyme and analyzed by thin-layer chromatography (Fig. 1). Ra-diolabeled material was released in a time-dependent fashion andmigrated slightly faster than the starting material under the chro-matography conditions used. The treatment of Mur-[14C]penta-peptide containing radiolabeled L-Ala gave identical results (datanot shown), suggesting that the enzyme cleaves between the L-Alaand the muramic acid residue or, although more unlikely, that theenzyme cleaves the pentapeptide at a position distal to the D-Gluresidue, a result that is consistent with the presence of an amidase2 domain, characteristic of N-acetylmuramoyl-L-alanine ami-dases identified by a search for conserved domains.

To unambiguously identify the site of cleavage, nonradiola-beled Mur-pentapeptide was treated with Ms6 LysA, and theresulting sample was purified by HPLC (3), using a Superdex Pep-tide 10/300 GL sizing column (Amersham Biosciences, Piscat-away, NJ), generating a single major peak consistent with a smallpeptide. Positive-ion mass spectroscopy showed a dominant fea-

Mycobacteriophage Ms6 LysA Is a Peptidoglycan Amidase

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ture with a retention time of 7.9 min and an m/z of 533.2, which isconsistent with a [M � H]� ion corresponding to a calculatedmonoisotopic mass of 532.249291 for the L-Ala–D-Glu–DAP–D-Ala–D-Ala pentapeptide. MS2 data confirmed this observation(Fig. 2; see also Fig. S1 in the supplemental material), demonstrat-ing the presence of daughter ions with m/z values of 515.18,462.07, 444.09, 373.05, and 333.04, as expected (3) (see Fig. S2 inthe supplemental material for the inferred fragmentation pat-tern). These results unambiguously show that Ms6 LysA is an

amidase that cleaves between the L-Ala residue and the lactyl moi-ety of the muramic acid residues of Mur-pentapeptide.

Effect of LysA on bacterial growth. Phage lytic enzymes oftenhave motifs resulting in tight and specific binding to the cell wall oftheir host bacterium, and exogenously applied phage-encoded en-dolysins have been shown to have effective antimicrobial activityagainst Gram-positive pathogens (18–20). Previously reportedobservations indicated that lysozyme could inhibit the growth ofMycobacterium bovis BCG and M. tuberculosis under some condi-tions (21, 22), and an MIC of 16 �g/ml for lysozyme against M.smegmatis grown in 7H9 medium was reported previously (23).Thus, a point that is not widely appreciated is that the mycolic acidlayer of mycobacteria, which is often described as a permeabilitybarrier (24–27), is not necessarily a barrier to lytic proteins. There-fore, the action of purified Ms6 LysA against intact mycobacteriawas explored even though the mycolic acid-rich cell wall is gener-ally thought to act as a permeability barrier. The addition of up to500 �g/ml of the endolysin neither inhibited the growth of norkilled M. smegmatis or M. tuberculosis in 7H9 medium. In con-trast, the treatment of 1.2 � 107 CFU M. smegmatis for 24 h withlysozyme at 20 �g/ml killed 98% of the bacteria (2.4 � 105 CFUremained), a result consistent with the low MIC of 16 �g/ml re-ported previously for M. smegmatis cells grown in 7H9 medium(23). It is not clear why an enzyme that cleaves the glycan chain ofpeptidoglycan, lysozyme, should kill mycobacteria when an en-zyme that removes the peptide portion of peptidoglycan, Ms6LysA, does not. It is possible that the explanation is trivial (insta-bility in the culture medium or a lack of high specific activity, for

FIG 1 TLC analysis of Mur-[14C]Glu-pentapeptide treated with purified re-combinant LysA for various periods of time. The radioactive material at theorigin is residual UDP-Mur-[14C]Glu-pentapeptide after hydrolysis to gener-ate Mur-[14C]Glu-pentapeptide.

FIG 2 MS2 of the pentapeptide L-Ala–�D-Glu–DAP–D-Ala–D-Ala released from Mur-pentapeptide by LysA. Observed ions are labeled according to the inferredfragmentation pattern shown in Fig. S2 in the supplemental material.

Mahapatra et al.

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example); however, phage lytic enzymes are generally ineffectiveagainst Gram-negative bacteria because the outer membranes ofthese organisms prevent access to the peptidoglycan, and the my-colic acid layer of the mycobacterial cell wall could act as a similar,although selective, barrier (12). It is possible that the considerablyhigher molecular mass of Ms6 LysA (43,079 Da) than that of ly-sozyme (16,240 Da) or other chemicophysical properties may pre-vent LysA from crossing the mycolate layer in intact mycobacteria.Alternatively, the polycationic properties of the lysozyme (28)may contribute to the molecule’s lethal effect on M. smegmatis;however, it is known that relatively minor changes in the mycolicacid structure result in a dramatic increase in the lysozyme sus-ceptibility of Mycobacterium marinum (29), suggesting that minorchanges in the physical nature of the mycolic acid layer can resultin significant changes in the permeability of this hydrophobic bar-rier. Recently, an intriguing idea that mycobacterial pathogensmight be rendered susceptible to exogenous endolysins throughcotreatment with LysA and LysB, a mycobacteriophage mycoly-larabinogalactan esterase that releases mycolic acids from the my-cobacterial cell wall, was proposed (17, 30).

Utilization of LysA for peptidoglycan analysis. Since it hadbeen demonstrated that Ms6 LysA efficiently solubilized the ma-jority of radiolabeled DAP in MAPc preparations (see above), itseemed likely that the enzyme could be utilized as a tool for anal-yses of the mycobacterial peptidoglycan structure. MS analysis ofMs6 LysA-treated MAPc from M. smegmatis identified 28 featureswith exact masses that matched the calculated masses of predictedpeptidoglycan fragments (Fig. 3). As can be seen, the solubilizedpeptides fell into one of three groups: those that were un-cross-linked monomers (Fig. 3) consisting of tri- and tetrapeptides;those that were cross-linked dimers of the stem peptide consistingof hexa-, hepta-, and octapeptides; and those that were cross-

linked trimers of the stem peptide consisting of nona- and deca-peptides. Thus, once incorporated into the cell wall, the entirerepertoire of detectable stem peptides in M. smegmatis peptidogly-can had been truncated to either tripeptides (Tri) (Fig. 3) or tet-rapeptides (Tetra) (Fig. 3). For example, features 7 and 9 are hexa-peptides composed of dimers of two stem peptides truncated totripeptides, with three carboxylic acid functions amidated (iden-tified as Tri-Tri-3NH2 in Fig. 3). Figure 4 provides a partial rep-resentative MS2 spectrum of a Tri-Tri-3NH2 peptide. This analysisclearly shows ions with m/z values (743.3666, 726.3424, and709.3167) consistent with the neutral loss of NH3, presumablyrepresenting the amide nitrogens (see Fig. S3 in the supplementalmaterial). Although these ions indicate the presence of the amidemodifications, they provide no information regarding the actuallocation of the solubilized peptidoglycan fragment.

The most abundant peptidoglycan fragment seen is a mono-mer with a tetrapeptide and 2 amide groups (feature 3), while thecross-linked dimer (Tri-Tri-2NH2) (feature 10) was the leastabundant (Fig. 3). In terms of cross-linking, it is obvious that theTri-Tri dimers must have DAP-DAP cross-links and that the Tri-Tri-Tri trimers must have two DAP-DAP cross-links. However,dimers and trimers containing a stem peptide with four aminoacid residues (Tetra) may have either a DAP-DAP or a DAP-Alacross-link. Interestingly, all of the trimers observed contain at leastone DAP-DAP cross-link (Fig. 3). The reason for this observationis not intuitively obvious; however, the most abundant monomeris Tetra (feature 3), and the most abundant dimer is Tetra-Tetra(feature 18). Since the most abundant fragments are Tetra frag-ments, they may well represent the substrates that are available forL,D-transpeptidation. The overall abundance of the Tri-Tri dimersand Tri-Tri-Tri trimers suggests the presence of an undescribedL,D-carboxypeptidase in M. smegmatis. While an activity of this

FIG 3 LC-MS analysis of LysA-treated MAPc from M. smegmatis. The molecular feature extractor algorithm (MFE) in Agilent MassHunter Qualitative Analysissoftware identified 28 features with exact masses that matched the calculated masses of predicted peptidoglycan fragments. These features are shown in a bubbleplot (A) and are listed in a table with retention times (RT), masses, peak heights (counts), and deduced structures (B).

Mycobacteriophage Ms6 LysA Is a Peptidoglycan Amidase

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nature was alluded to in a recent review (31), there appear to be noempirical data describing the activity available.

Degree of amidation in peptidoglycan of M. smegmatis. Cellwalls were prepared from M. smegmatis and treated with Ms6LysA, and the solubilized material was analyzed as describedabove. The observed peptides ranging from tripeptide monomersto decapeptide trimers have between 3 and 7 carboxylic acid resi-dues that potentially could be amidated. In all cases observed, atleast one of the carboxylic acid residues was amidated (Fig. 3). Thetripeptide monomers had two amidated carboxylic acids residuesin at least two configurations (as determined by relative retentiontimes during chromatography), as did the tetrapeptide mono-mers. The hexa-, hepta-, and octapeptides had predominantly 3modified carboxylic acid residues. In contrast, the nona- and de-capeptides had primarily 4 or 5 modified carboxylic acid residues,although there were a few with only three modified acidic resi-dues. Hence, the amidation previously found at the lipid II level(3, 4) carried over to mature peptidoglycan, which is heavily mod-ified. The significance of this modification is unknown at present,but one could speculate that the reduction of polarity could makeit more energetically favorable to move the lipid II across theplasma membrane; however, all bacteria with peptidoglycantransport lipid II across the plasma membrane, but amidation isnot a feature of the peptidoglycan of all bacteria. Alternatively, theamide modifications could help regulate the degree and nature ofthe interpeptide cross-linking via an as-yet-unknown mechanism,they could play a role in the coordination of peptidoglycan assem-bly with that of other components of the cell wall, or they couldprovide resistance to the activity of exogenous lytic enzymes. Theidentification of the precise positions of carboxylic acid residuesthat are amidated could potentially shed light on this issue.

Conclusions. We have shown that LysA from phage Ms6 is a

peptidoglycan amidase that cleaves the bond between the D-Murand L-Ala of the stem peptide of Mur-pentapeptide and maturepeptidoglycan. Unlike lysozyme, the Ms6 LysA enzyme was un-able to kill or inhibit the growth of either M. smegmatis or M.tuberculosis under the conditions tested. However, the enzyme hasbeen shown to be useful for solubilizing the peptide chains ofmycobacterial peptidoglycan for further analysis. In addition, itwas shown that the mature peptidoglycan of M. smegmatis ishighly amidated, similar to the amidation previously reported forthe peptidoglycan precursor lipid II.

ACKNOWLEDGMENTS

This work was supported by funds provided by U.S. Public Health Servicegrants NIH/NIAID AI-33706 (M.R.M.), NIH/NIAID AI049151 (D.C.C.),and NIH/NIAID AI073772 (M.S.P.) and FCT (Fundação para a Ciência eTecnologia) project POCTI/ESP/47723/2002 (J.M.-P. and M.P.).

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